The venom of the red imported fire ant, Solenopsis invicta, is dominated by trans stereoisomers of 2,6-dialkylpiperidines. cis Stereoisomers of alkaloids in the venom of S. invicta were separated from trans stereoisomers by using silica gel short column chromatography and identified by coupled gas chromatography mass spectrometry (GC-MS). Seven pairs of cis and trans sterecoisomers were identified based on relative retention times and mass spectral data. The GC trace of the cis stereoisomers of S. invicta alkaloids was presented for the first time. In addition to the previously described 2,6-dialkylpiperideines, eleven novel 2,6-dialkyl-delta1,2-piperideines and 2,6-dialkyl-delta1,6-piperideines were identified from S. invicta venom. The results are discussed in relation to the evolutionary significance of these piperideines and their possible biosynthetic pathways in Solenopsis fire ants.